Composition for external use on skin for inflammatory diseases

ABSTRACT

[Problem] To provide a composition for external skin use that has anti-inflammatory and antiallergic activity, and is very safe. [Solution] A composition for external skin use for inflammatory diseases contains dihomo-gamma-linolenic acid (DGLA) as the active ingredient thereof. It is preferable for the DGLA to be present as a glyceride, a phospholipid, or an alkyl ester. The amount of DGLA contained in the composition for external use is 0.1-50 wt %.

TECHNICAL FIELD

The present invention relates to a composition for external use on skinthat has therapeutic effect on various kinds of inflammatory diseasesuch as dermatitis such as atopic dermatitis, contact dermatitis orUV-caused skin disorder, and rheumatism, psoriasis and lupus.

BACKGROUND ART

As our living environment is changing or becoming complex or with recentchanges in our food life, cases of allergic skin disease are rapidlyincreasing in number. Exemplary allergic skin diseases are atopicdermatitis and contact dermatitis. Various methods are currentlyemployed to treat these allergic skin diseases and they includeavoidance of allergens through a better living environment or a dietarytherapy and the administration of anti-allergic agents or steroids. Asof today, however, no completely effective method has yet beenestablished to treat allergic skin diseases and a therapeutic drug isdesired that is safe to use without developing any side effects andwhich is capable of exhibiting satisfactory therapeutic efficacy.

Dihomo-γ-linolenic acid (8,11,14-eicosatrienoic acid), which ishereinafter abbreviated as DGLA, is an n-6 polyunsaturated fatty acidcontained in breast milk, fish oil, seaweeds, meats, etc. and may besynthesized in vivo from linolic acid via γ-linolenic acid. Heretofore,the physiological actions of the orally administered DGLA precursorγ-linolenic acid have been studied to yield numerous reports concerninghypertension, arterial infarction, complications of diabetes mellitus(neuropathy), and rheumatism, and a lot of cases have also been reportedconcerning diseases such as atopic dermatitis that involve allergicinflammations (for example, Non-Patent Document 1). A possibleanti-inflammatory mechanism of orally administered γ-linolenic acid isspeculated to start with conversion of γ-linolenic acid to DGLA which inturn is metabolized by cyclooxigenase or 15-lipoxygenase intoprostaglandin E1 (PGE1) which competes with prostaglandin E2 or into15-hydroxyeicosatrienoic acid (15-HETrE) which inhibits 5-lipoxygenase.As for DGLA, partly because of the lack of a process for its massproduction, fewer clinical reports have been published than forγ-linolenic acid. Recently, however, a microorganism-based technologyfor DGLA production was discovered (Patent Document 1) and an atopy-likedermatitis suppressing action (Patent Document 2) and an anti-arterialinfarction action have been reported from experiments with animalmodels. In those experiments, however, the feed was a mixed dietcontaining a DGLA oil which was administered orally.

Few studies have been reported on the use of γ-linolenic acid or DGLA asagents for external use, and in the single clinical report of the casewhere a cream using a γ-linolenic acid containing oil was used inpatients with atopic dermatitis, some improvement was recognized in aself-appraisal by the patients themselves but no significant differencewas recognized in a doctor's appraisal (Non-Patent Document 2.)

Patent Documents 3 and 4 suggest that essential fatty acids includingDGLA be used as agents for external use for skin disease but no workingexamples were given. Patent Document 5 discloses the COX-2 inhibitoryaction of DGLA and suggests that it is used in inflammatory skindisease.

CITATION LIST Patent Documents

-   Patent Document 1: JP 3354581-   Patent Document 2: WO 2006/085687-   Patent Document 3: EP 0173478-   Patent Document 4: EP 0085579-   Patent Document 5: WO 2010/125340

Non-Patent Documents

-   Non-Patent Document 1: Current Pharmaceutical Biotechnology, 7,    p503-524, 2006. “A Meta-Analysis of Randomized, Placebo-Controlled    Clinical Trials of Efamol (Trade Mark) Evening Primrose Oil in    Atopic Eczema. Where Do We Go from Here in Light of More Recent    Discoveries?”-   Non-Patent Document 2: J. Dermatological Treatment, 1, p199-201,    1990, “Topical evening primrose oil as treatment for atopic eczema.”

SUMMARY OF INVENTION Technical Problem

The object of the present invention is to provide a composition forexternal use on skin that is safe to use and has an anti-inflammatoryaction. Drugs of first choice currently used to treat chronic skindiseases such as atopic dermatitis are agents for external use such assteroids and the immunosuppressant FK-506 (common name: tacrolimus;trade name: Protopic). Although showing a potent anti-inflammatoryaction, these drugs are suspected to cause side effects, so they aredirectly administered to the diseased area as an agent for external useto reduce the possibility of systemic side effects. Nevertheless, thesedrugs still have side effects and the advent of a safer agent forexternal use is desired.

The term composition for external use as used herein means compositionsthat are to be directly applied to the skin, as exemplified bypharmaceutical drugs for external use, quasi-drugs for external use, andcosmetic products.

The “pharmaceutical drugs” are intended for use in diagnosis, treatmentor prevention of diseases in humans or animals; the “cosmetic products”are intended for use on the human body by rubbing, spraying or likemethods in order to keep it clean, beautify it, make it more attractive,change the facial appearance, or keep the skin or hair in healthycondition, provided that they exert a mild action on the human body; andthe “quasi-drugs” which are intermediate between the two categories havemore distinct efficacy than cosmetic products and prevent skin troubleswithout causing side effects. In countries where no such definitions areavailable, the composition for external use of the present inventionshall be understood in the context of equivalents thereto.

Solution to Problem

Although DGLA has been reported to be effective against skin disease ifit is ingested by oral administration, there is no report on the effectthat is obtained by using DGLA as an agent for external use on skin.Since highly unsaturated fatty acids including γ-linolenic acid andeicosapentaenoic acid serve as the substrates for enzymes includingcyclooxygenase and lipoxygenase which are involved in inflammations andallergy, they have been studied for their possible involvement ininflammations and allergic disease but their effects are so mild thatonly a few have reached the level of development as pharmaceuticaldrugs.

While making studies of safer and more efficacious compositions forexternal use, the present inventors found that DGLA showed anunexpectedly potent action against atopic dermatitis and this findinghas led to the accomplishment of the present invention.

The gist of the present invention comprises compositions for externaluse on skin as recited in (1) to (9) below:

(1) A composition for external use on skin in the treatment ofinflammatory disease which comprises dihomo-γ-linolenic acid (DGLA) asan active ingredient.(2) The composition for external use on skin in the treatment ofinflammatory disease as recited in (1), wherein the dihomo-γ-linolenicacid (DGLA) is contained in the form of any one of a glyceride, aphospholipid, a glycolipid, an alkyl ester, a salt, or a free fattyacid.(3) The composition for external use on skin in the treatment ofinflammatory disease as recited in (2), wherein the glyceride is atriglyceride, a diglyceride, or a monoglyceride.(4) The composition for external use on skin in the treatment ofinflammatory disease as recited in (2), wherein the alkyl ester is anester with a lower C₁₋₄ alcohol.(5) The composition for external use on skin in the treatment ofinflammatory disease as recited in (2), wherein the phospholipid isdiacylglycerophospholipid or lysoacylglycerophospholipid.(6) The composition for external use on skin in the treatment ofinflammatory disease as recited in any one of (1) to (5), which contains0.01 to 50 wt % of the dihomo-γ-linolenic acid (DGLA).(7) The composition for external use on skin in the treatment ofinflammatory disease as recited in any one of (1) to (6), wherein theinflammatory disease is an inflammatory skin disease.(8) The composition for external use on skin in the treatment ofinflammatory disease as recited in (7), wherein the inflammatory diseaseis atopic dermatitis, allergic contact dermatitis, irritant contactdermatitis, psoriasis, rheumatism, or lupus.(9) The composition for external use on skin as recited in any one of(1) to (8), which is a formulation that contains 0.01 to 50 wt % of thedihomo-γ-linolenic acid (DGLA) and is applied continuously for at least7 consecutive days to an area affected by the inflammatory disease.(10) The composition for external use on skin as recited in (9), whichis a formulation that contains 0.05 to 10 wt % of the dihomo-γ-linolenicacid (DGLA) and is applied continuously for at least 7 consecutive daysto an area affected by the inflammatory disease.(11) The composition for external use on skin as recited in (9), whichis a formulation that contains 0.05 to 7 wt % of the dihomo-γ-linolenicacid (DGLA) and is applied continuously for at least 7 consecutive daysto an area affected by the inflammatory disease.(12) The composition for external use on skin as recited in (9), whichis a formulation that contains 0.01 to 50 wt % of the dihomo-γ-linolenicacid (DGLA) and is applied continuously for at least 10 consecutive daysto an area affected by the inflammatory disease.(13) The composition for external use on skin as recited in (9), whichis a formulation that contains 0.01 to 50 wt % of the dihomo-γ-linolenicacid (DGLA) and is applied continuously for at least 14 consecutive daysto an area affected by the inflammatory disease.(14) The composition for external use on skin as recited in any one of(1) to (13), which is for suppressing the itch of the inflammatorydisease.

Advantageous Effects of Invention

When applied to the skin, the agent for external use of the presentinvention has therapeutic effect on an inflammatory skin disease orallergic skin disease, as exemplified by atopic dermatitis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the results of the skin irritation testconducted in Example 3.

FIG. 2 is a graph showing the changes in clinical symptom scores asobserved in Example 4, the data being indicated by the mean±S.E. formice in groups each consisting of 7 animals. *: p<0.05, Wilcoxon signedrank test.

FIG. 3 graphically shows the changes in scratching behavior (scratchingfrequency and duration in 20 minutes) as observed in Example 4, the databeing indicated by the mean±S.E. for mice in groups each consisting of 7animals.

FIG. 4 is a graph showing the changes in body weight as observed inExample 4, the data being indicated by the mean±S.E. for mice in groupseach consisting of 7 animals.

FIG. 5 is a graph showing the changes in clinical symptom scores asobserved in Example 5, the data being indicated by the mean±S.E. formice in groups each consisting of 7 animals.

FIG. 6 graphically shows the changes in scratching behavior (scratchingfrequency and duration in 30 minutes) as observed in Example 5, the databeing indicated by the mean±S.E. for mice in groups each consisting of 7animals. *: p<0.05, Kruscal-Wallis test, Dunn's multiple comparisontest.

FIG. 7 is a graph showing the changes in clinical symptom scores asobserved in Example 6, the data being indicated by the mean±S.E. formice in groups each consisting of 7 animals. *: p<0.05, **: p<0.01, ***:p<0.001, Kruscal-Wallis test, Dunn's multiple comparison test.

FIG. 8 graphically shows the changes in scratching behavior (scratchingfrequency and duration in 30 minutes) as observed in Example 6, the databeing indicated by the mean±S.E. for mice in groups each consisting of 7animals. *: p<0.05, Kruscal-Wallis test, Dunn's multiple comparisontest.

FIG. 9 is a graph showing the changes in transepidermal water loss asobserved in Example 6, the data being indicated by the mean±S.E. formice in groups each consisting of 7 animals. *: p<0.05, Kruscal-Wallistest, Dunn's multiple comparison test.

DESCRIPTION OF EMBODIMENTS

The dihomo-γ-linolenic acid (DGLA) as used in the present invention isan n-6 unsaturated fatty acid with a 20-carbon chain and three doublebonds. The DGLA to be used for the purpose of the present invention maytake on any form that contains it as a constituent fatty acid andexamples include a free fatty acid, salts such as sodium and potassium,lower C₁₋₄ alkyl esters such as ethyl ester and methyl ester, glycerinesters such as triglyceride, diglyceride, and monoglyceride,phospholipids such as diacylglycerophospholipid andlysoacylglycerophospholipid, and glycolipids. Preferred are esters suchas glycerin esters and lower alkyl esters, with ethyl ester beingparticularly preferred.

DGLA can be mass-produced by the method publicly disclosed in JapanesePatent No. 3354581. Specifically, a microorganism of the genusMortierella capable of producing a DGLA-containing oil is cultured andthe oil is extracted from the cultured cells. The oil as obtained bythis method contains 30-50% of DGLA, so it may be immediately used;alternatively, it may be used after enriching the DGLA. Specifically,the DGLA containing oil is subjected to wintering, treatment with anenzyme such as lipase, or the like to enrich the DGLA. In anotherprocess, the DGLA containing oil is subjected to hydrolysis oralcoholysis to effect conversion to the free fatty acid, a salt thereof,an ester thereof with a lower alcohol, or the like. If desired, furtherenrichment may be performed by distillation, urea adduct process, columntreatment, enzymatic treatment, supercritical carbon dioxide treatment,or the like.

The pharmaceutical drug for external use, quasi-drug for external use,and the cosmetic product that are provided by the present invention maytake on any dosage form that enables the active ingredient to bedirectly administered to the topical surface of the skin. Examplesinclude ointments such as oleaginous ointments, emulsion ointments(creams) and water-soluble ointments; as well as gels, lotions, tapes,patches, sprays, gels, and reservoir-type patches.

Depending on the need, the composition for external use of the presentinvention permits the use of any materials that are commonly used incompositions for external use and they include bases, absorptionpromoters, moisturizers, thickeners, emulsifying agents, colorants,fragrances, antioxidants, stabilizers, sterilizers, and antiseptics.

Bases for external use include, for example, white petrolatum, cetanol,stearyl alcohol, stearic acid, white beeswax, liquid paraffin,lauromacrogol, squalane, squalene, lanolin, isobutyl myristate, andmiddle-chain fatty acid triglyceride. Since the highly unsaturated fattyacids or their salts or esters are oleaginous, they can be convenientlymixed with oleaginous bases to form a homogeneous mixture.Water-containing bases can also be used if they are emulsions (W/O orO/W type) and the active ingredient can be readily mixed byincorporating it into the oil phase.

Examples of the absorption promoter include urea, crotamiton, diethylsebacate, diisopropyl adiptate, ethanol, isopropyl alcohol, dimethylsulfoxide, 1-dodecylazacycloheptan-2-one, 1-menthol, salicylic acid,pyrrolidones, middle-chain fatty acid esters, phosphatidylcholine,silicone-based promoters, and limonene.

Exemplary moisturizers include polyhydric alcohols such as glycerin,sorbitol, propylene glycol, 1,3-butanediol, sodium pyrrolidonecarboxylic acid, and sodium hyaluronate.

Exemplary thickeners include gum arabic, guar gum, locust bean gum,carrageenan, carboxymethylcellulose, carboxymethylcellulose sodium,polyacrylic acid salts, polyacrylic acid esters, natural latex, vinylacetate resin emulsion, polyvinyl alcohol, hydroxypropyl cellulose, andhydroxypropyl methylcellulose.

Exemplary emulsifying agents include glycerin fatty acid esters, sucrosefatty acid esters (sugar esters), sorbitan fatty acid esters, propyleneglycol fatty acid esters, lecithin, polyoxyethylene fatty acid esters,polyloxyethylene castor oil, polyoxyethylene hardened castor oil,polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acidesters, and sodium lauryl sulfate.

Exemplary fragrances include eucalyptus oil, lavender oil, menthol,peppermint oil, rose oil, orange oil, cherry flavor, fruit flavor,vanillin, and vanilla flavor.

Exemplary antioxidants include L-ascorbic acid, ascorbyl stearate,ascorbyl palmitate, sodium ascorbate, erythorbic acid, sodiumerythorbate, isopropyl citrate, dl-α-tocopherol, dl-δ-tocopherol,dibutylhydroxytoluene (BHT), and butylhydroxyanisole (BHA). Among these,ascorbyl palmitate, ascorbyl stearate, L-ascorbic acid, dl-α-tocopherol,dl-δ-tocopherol, or combinations thereof are preferred.

Exemplary stabilizers include polysorbates, polyethylene glycol,ethanol, acetone, light silicic anhydride, and EDTA.

Exemplary antiseptics include parahydroxybenzoic acid esters,benzalkonium chloride, sorbic acid, phenol, chlorobutanol, chlorocresol,and benzyl alcohol.

The composition for external use of the present invention mayadditionally contain other active principles, such as steroids,anti-allergic agents, immunosuppressants, and anti-inflammatory drugs,which are commonly used for the treatment of inflammatory diseasesincluding various types of dermatitis, rheumatism, psoriasis, and lupus.

The composition for external use of the present invention is usefulagainst inflammatory diseases including various types of dermatitis,rheumatism, and psoriasis that have been shown to be effectively treatedwith highly unsaturated fatty acids. Stated specifically, thecomposition can be directly applied to areas affected by atopicdermatitis, allergic contact dermatitis, irritant contact dermatitis,rheumatism, psoriasis, and lupus.

The amount in which the composition for external use of the presentinvention is to be used is variable over a wide range depending uponsuch factors as the symptom, the surface area of the region affected bythe disease, the age and body weight of the patient, and the mode ofdrug administration; the general standard dosage is within the rangefrom 0.1 to 5 g per day and this is directly applied to the diseasedarea once or several times a day.

The concentration of DGLA to be contained in the composition forexternal use preferably ranges from about 0.01 to about 50 wt %, morepreferably from 0.05 to 10 wt % or from 0.05 to 7 wt %. Depending on therelative content of DGLA in DGLA containing oil, a suitable amount ofthe DGLA containing oil may be mixed in the composition for externaluse. Since the DGLA itself is in oil form, the required amount of it canbe readily mixed in any base for agents for external use.

As will be shown by the results of Example 4 (FIG. 2), the agent forexternal use on skin of the present invention exhibits a comparablelevel of efficacy to the Protopic ointment which is currently used as atherapeutic agent for atopic dermatitis. A recommended method of usingthe agent involves continuous application for at least 7 days,preferably at least 10 days, more preferably at least 14 days. When thisagent was administered for 3 weeks, the clinical symptom score of atopicdermatitis could be lowered to a level comparable to that achieved bythe Protopic ointment. The continuous application may be substantiallycontinuous or continuous with an interval of one or two days asexemplified by a two-week application, with 5 days per week.

The followings show exemplary formulations for the composition forexternal use of the present invention. The concentrations of the DGLAcontaining oil that are indicated in the tables may be increased ordecreased as appropriate.

(1) Oleaginous Base

[White ointment]

TABLE 1 Ingredient Content (in 100 g) DGLA containing oil 5.0 g Whitebeeswax 4.75 g  Sorbitan sesquioleate 1.9 g White petrolatum bal.

[White Petrolatum]

TABLE 2 Ingredient Content (in 100 g) DGLA containing oil  5.0 g Whitepetrolatum 95.0 g

(2) Emulsion base

[Hydrophilic Ointment]

TABLE 3 Ingredient Content (in 100 g) DGLA containing oil 5.0 g Whitepetrolatum 23.75 g Stearyl alcohol 19.0 g Propylene glycol 11.4 gPolyoxyethylene hardened castor oil 60 3.8 g Glyceryl monostearate 0.95g Methyl parahydroxybenzoate 0.095 g Propyl parahydroxybenzoate 0.095 gPurified water bal.

[Purified Lanolin]

TABLE 4 Ingredient Content (in 100 g) DGLA containing oil  5.0 gPurified lanolin 95.0 g

[Absorptive Ointment]

TABLE 5 Ingredient Content (in 100 g) DGLA containing oil 5.0 g Whitepetrolatum 38.0 g Cetyl alcohol 9.5 g White beeswax 4.75 g Sorbitansesquioleate 4.75 g Lauromacrogol 0.475 g Methyl parahydroxybenzoate0.095 g Propyl paraohydroxybenzoate 0.095 g Purified water bal.

(3) Zinc Oxide Ointment

TABLE 6 Ingredient Content (in 100 g) DGLA containing oil 5.0 g Zincoxide 19.0 g Liquid paraffin 2.85 g White ointment bal.

(4) Urea Cream

TABLE 7 Ingredient Content (in 100 g) DGLA containing oil 5.0 g Urea4.75 g Cetyl alcohol 4.75 g Glyceryl monostearate 4.75 g Liquid paraffin4.75 g Glycerin 9.5 g Polyoxyethylene(20) cetyl ether 2.375 gMethylpolysiloxane 0.95 g Methyl parahydroxybenzoate 0.19 g Tetrasodiumedetate 0.19 g Sodium citrate 0.19 g Purified water bal.

On the following pages, working examples of the present invention areset out but they are by no means intended to limit the scope of thepresent invention.

Unless otherwise noted, all indications of percentages in the workingexamples are on a weight basis.

Example 1

Preparation of DGLA Ethyl Ester (DGLA-EE) and DGLA ContainingTriglyceride (DGLA-TG)

A DGLA containing oil was obtained by the method described in U.S. Pat.No. 3,354,581. Specifically, 500 mL of hexane was added to 200 g ofdried cells of a filamentous fungus (Mortierella alpine) and the mixturewas stirred and filtered; by repeating these steps three times, an oilfraction was extracted and the hexane layer was evaporated to give aDGLA containing oil.

The resulting DGLA containing oil was analyzed for the fatty acidcomposition. A portion (25 μL) of the oil was collected in a test tubeand after adding 1.5 mL of a 0.5N aqueous solution of sodium hydroxide,the mixture was stirred well while it was heated at 80° C. for 5 minutesin a nitrogen gas stream. Thereafter, 2 mL of a reagent solutionconsisting of 14% boron trifluoride in methanol was added and themixture was stirred well while it was heated at 80° C. for 80 minutes ina nitrogen gas stream. After the heating, the mixture was air-cooled toapproximately between 30° C. and 40° C. and following the addition ofisooctane (2 mL), the mixture was vigorously agitated for one minute ina nitrogen gas stream; thereafter, 5 mL of saturated saline was addedand the mixture was allowed to stand at room temperature. The separatingisooctane layer was recovered and used as a sample in gas chromatographyto analyze the fatty acid composition. Indicated below are theconditions for measurement by gas chromatography. The results are shownin Table 8; the values in Table 8 refer to the proportions (%) of therespective fatty acids, with the total quantity of the fatty acids beingtaken as 100%. The DGLA containing oil was found to contain about 39.8area % of DGLA.

<Conditions for Measurement by Gas Chromatography>

Apparatus: 6890N Network GC System (Agilent)

Column: DB-WAX (Agilent J&W); 30 m (length)×0.250 mm (insidediameter)×0.25 μm (membrane thickness)Column temperature: initial temperature, 180° C.; temperature elevation,3° C./min; final temperature, 230° C.; retained for 15 minutesInjection port temperature: 250° C.Detector temperature: 250° C.

Detector: FID

Carrier gas: helium

TABLE 8 Fatty Acid Composition in DGLA Containing Oil Fatty acidComposition (%) C16:0 16.1 C18:0 7.3 C18:1 n-9 8.5 C18:2 n-6 6.7 C18:3n-6 2.9 C18:3 n-3 0.6 C20:0 0.8 C20:3 n-6 39.8 C20:4 n-6 0.7 C22:0 2.6C24:0 9.2

[Preparation of DGLA-EE]

An egg-plant shaped flask was charged with 15 g of the DGLA containingoil, 5 mL of ethanol and 1.73 mL of 20% sodium ethoxide/ethanol, and themixture was refluxed in an oil bath (115° C.) for 120 minutes. Thereaction mixture was distributed between hexane and water and the hexanelayer was evaporated to give 15.4 g of a crude product. The crudeproduct was fractionated with a silica gel column (eluent: hexane/ethylacetate=15:1) and after collecting the ethyl ester fractions, thesolvent was distilled off to give 11.4 g of DGLA-EE. After adding 0.5%(w/w) of δ-tocopherol, the product was used as DGLA-EE in the followingworking examples.

[Preparation of DGLA-TG]

Fifteen grams of the DGLA containing oil was fractionated with a silicagel column (eluent:hexane:ethyl acetate=10:1) and after collecting thetriglyceride fractions, the solvent was distilled off to give 12.5 g ofDGLA-TG. After adding 0.5% (w/w) of δ-tocopherol, the product was usedas DGLA-TG in the following working examples.

Example 2

Preparation of DGLA Ointments

The above-mentioned DGLA-EE or DGLA-TG (10 wt %), white petrolatum(89.95 wt %), and ascorbyl palmitate (0.05 wt %) were mixed underheating at 60° C. to prepare two kinds of DGLA containing ointment. Theconcentration of DGLA in each ointment was about 4%. A mixture of whitepetrolatum (99.95 wt %) and ascorbyl palmitate (0.05 wt %) was used as anegative control.

Example 3

Skin Irritation Test

To the left auricle of each 6-week old, male ddy mice, a suitable amountof the base (negative control) was applied whereas a suitable amount ofthe DGLA-EE containing ointment or the DGLA-TG containing ointment wasapplied to the right auricle of the same individual; both applicationswere conducted once daily for 5 consecutive days; thereafter, thethickness of each auricle was measured and its state was observed (n=6).In addition, 100 mg of each ointment was applied to the back of 6-weekold, male ddy mice once daily for 5 consecutive days and the state ofthe dorsal skin was observed (n=2). As a result, as shown in FIG. 1, nosignificant change was recognized in either group as regards thethickness of auricle. The states of each auricle and the dorsal skinwere observed and inflammation or the like was not recognized. Thesefindings led to the conclusion that both the DGLA-TG containing ointmentand the DGLA-EE containing ointment would be highly safe compositionsfor external use.

Example 4

Effect on Atopic Dermatitis Model Mice

Using NC/Nga mice, which served as a model for spontaneous atopicdermatitis, the anti-inflammatory action of the DGLA containingointments was examined.

[Test Method]

Four groups (7 animals per a group) of NC/Nga mice that hadspontaneously developed symptoms of atopic dermatitis were provided andto the respective groups, the DGLA-TG containing ointment, the DGLA-EEcontaining ointment, the Protopic ointment as the positive control(product of Astellas Pharma Inc.; containing 0.1% tacrolimus), and thebase as the negative control were applied and the resulting changes indermal symptoms were evaluated in scores. In addition, the effects onscratching behavior as an index of itch were quantitatively analyzedwith SCLABA-Real® (Noveltec Inc., Kobe).

[Grouping]

The animals that developed erythema, edema and scabbing on the neck anddorsal part during the period of acclimatization were carefully shavenover the area extending from the back of the head through the back ofthe neck to the dorsal part and those animals that had moderate symptomsof dermatitis such as erythema, edema and scabbing in the neck or thedorsal part were selected at intervals of several days and grouped insuch a way that the clinical symptom scores would be as uniform aspossible among the groups.

[Administration of Test Substances]

To the shaven neck and dorsal part, about 100 mg of each test substancewas applied once daily for three weeks on a 5-days-a-week basis. Eachapplication was conducted every day from 10:00 to 12:00.

[Items of Observation]

The symptomatic state of the outer surface of the site to which eachtest substance had been applied was observed before the test, twice aweek during the administration, and the day after the final application,and the clinical symptom scores of the symptoms on the neck and dorsalpart were recorded based upon the following descriptions ofclassification.

(1) Symptom scoring criteria (neck and dorsal part score criteria)

1) Items of Observation

-   -   pruritus/itching    -   erythema/hemorrhage    -   edema    -   excoriation/erosion    -   scaling/dryness

2) Grading

The above-mentioned items of observation were graded by the scores givenbelow.

0 No Symptoms 1 Light 2 Moderate 3 Severe

(2) Body Weight Measurement

Body weight measurement was conducted the day before the start ofapplication, as well as at days 8, 15 and 22 thereafter (day 22corresponding to the day after final application).

(3) Scratch Counting

The day before the start of application and at day 22 thereafter (theday after final application), scratch counting was performed. Scratchingfrequency was measured for 20 minutes using an automatic scratchinganalyzing system (SCLABA-Real® of Noveltec).

[Results]

(1) Changes in Clinical Symptom Scores (FIG. 2)

The clinical symptom scores prior to the test were 6.4-6.9 on average ineach group (moderate manifestation), with no statistical differencerecognized between groups. In the DGLA-TG and DGLA-EE groups, theclinical symptom scores started to improve from the second to thirdweeks and, in particular, the dermatitis score for the fifth evaluation(at week 3) to the DGLA-EE group showed a statistically significant dropcompared to the score before the start of the test (pre) (p<0.05,Wilcoxon signed rank test). In the Protopic group, the improvementtended to occur earlier, from the first to second weeks. In any event,at the time the test ended, there was no difference in dermatitis scoresamong the three groups, the DGLA-TG group, the DGLA-EE group, and theProtopic group, and they were recognized to be comparable in dermatitissuppressing action

(2) Changes in Scratching Behavior (FIG. 3)

The frequency and duration of scratching behavior were measured for 20minutes before the start of the test and no statistical difference wasrecognized between groups. When measured after the end of the test, thefrequency and duration of scratching behavior tended to decrease in eachof the DGLA-TG group, the DGLA-EE group, and the Protopic group, but nostatistical difference was recognized.

(3) Changes in Body Weight (FIG. 4)

Throughout the test period, no statistically marked difference wasrecognized between groups as regards the average body weight.

As shown above, both the DGLA-TG containing ointment and the DGLA-EEcontaining ointment had an immunosuppressive effect, which was by nomeans inferior to the Protopic ointment which is currently used as thedrug of first choice against atopic dermatitis and the like.

Example 5

A test was conducted with atopic dermatitis model mice to compare theeffect of the ethyl ester type DGLA-EE with that of the free type DGLA.The test method was essentially the same as described in Example 4;about 100 mg of each test substance was applied to the shaven neck anddorsal part once daily for 4 weeks on a five-days-a-week basis.

Preparation of DGLA Containing Ointments

A DGLA-EE or DGLA containing ointment was prepared by mixing DGLA-EE orDGLA with white petrolatum as in Example 2 so that the concentration ofDGLA-EE or DGLA would be about 10%. Note that DGLA was obtained byhydrolyzing DGLA-EE with potassium hydroxide.

[Results]

(1) Changes in Clinical Symptom Scores (FIG. 5)

The clinical symptom scores prior to the test were 5.1-5.7 on average ineach group (moderate manifestation), with no statistical differencerecognized between groups. Four weeks later, the symptoms of the basegroup worsened as indicated by a score of 7.9, whereas the symptom scoreof the DGLA-EE ointment group tended to improve, as indicated by thevalue of 4.0. The DGLA ointment group showed a slight tendency toimprove as compared with the base group, whereas the efficacy was not asgreat as in the DGLA-EE ointment group. It is therefore concluded thatDGLA as an agent for external use preferably takes on the form of anethyl ester.

(2) Changes in Scratching Behavior (FIG. 6)

The frequency of scratching behavior was measured within a period of 30minutes before the start of the test and no statistical difference wasrecognized between groups. When measured after 4 weeks following thestart of the test, both the DGLA-EE ointment group and the DGLA ointmentgroup tended to improve the frequency of scratching behavior as comparedwith the base group and, particularly in the DGLA-EE ointment group, thefrequency of scratching behavior decreased to 13.3 on average ascompared with 48.6 for the base group. As for the duration of scratchingbehavior that was measured within a period of 30 minutes, no statisticaldifference was recognized between groups before the start of the test;when the test ended 4 weeks later, however, both the DGLA-EE ointmentgroup and the DGLA ointment group had been improved in the duration ofscratching behavior and, particularly in the DGLA-EE ointment group, theduration of scratching behavior at week 3 was 4.5 seconds, which was asignificant decrease from the value of 32.5 seconds for the base group(p<0.05, Kruscal-Wallis test, Dunn's multiple comparison test).

Example 6

A test was conducted with atopic dermatitis model mice to study the dosedependency of the DGLA-EE containing ointment. The test method wasessentially the same as described in Example 4, except that thetransepidermal water loss (TEWL) was an additional item of measurement;about 100 mg of the test substance was applied to the shaven neck anddorsal part once daily for 4 weeks on a five-days-a-week basis.

Preparation of DGLA Ointments

DGLA-EE containing ointments were prepared by mixing DGLA-EE with whitepetrolatum so that the concentration of DGLA-EE would be 5, 10, and 20%.

[Item of Observation]

The transepidermal water loss (TEWL) was measured before the start ofthe test as well as at weeks 2 and 4. The measurement was conducted witha multi probe adapter (CK Electronic GmbH, Germany); the sensing probewas pressed against the site on the neck and dorsal part skin where thetest substance had been applied and a substantially uniform pressure wasmaintained until the reading stabilized; three measurements wereconducted and their average was taken.

[Results]

(1) Changes in Clinical Symptom Scores (FIG. 7)

The clinical symptom scores prior to the test were 7.0-7.3 on average ineach group (moderate manifestation), with no statistical differencerecognized between groups. Four weeks later, the symptoms of the basegroup worsened as indicated by a score of 10.0, whereas the symptomscores of the DGLA-EE groups improved in a dose-dependent manner. Inparticular, the score of the 5% DGLA-E ointment group significantlydecreased to 3.9 as compared with the base group (p<0.01, Kruscal-Wallistest, Dunn's multiple comparison test).

(2) Changes in Scratching Behavior (FIG. 8)

The frequency of scratching behavior was measured within a period of 30minutes before the start of the test and no statistical difference wasrecognized between groups. When measured after 4 weeks following thestart of the test, each of the DGLA-EE ointment groups tended to improvethe frequency of scratching behavior as compared with the base group ina dose-dependent manner and, particularly in the 5% DGLA-EE ointmentgroup, the frequency of scratching behavior significantly decreased to6.0 as compared with the value of 67.0 for the base group (p<0.05,Kruscal-Wallis test, Dunn's multiple comparison test). As for theduration of scratching behavior that was measured within a period of 30minutes, no statistical difference was recognized between groups beforethe start of the test; when the test ended 4 weeks later, however, eachof the DGLA-EE ointment groups had improved the duration of scratchingbehavior in a dose-dependent manner and, particularly in the 5% DGLA-EEointment group, the duration of scratching behavior was 3.9 seconds,which was a significant decrease from 33.1 seconds for the base group(p<0.05, Kruscal-Wallis test, Dunn's multiple comparison test).

(3) Transepidermal Water Loss (FIG. 9)

In the 5% DGLA-EE ointment group and the 10% DGLA-EE ointment group,TEWL was 27.8 and 31.1 g/hr/m², respectively, before the start of thetest; after 4 weeks following the start of the test, however, theysignificantly dropped to 11.5 and 16.5 g/hr/m² (p<0.05, Kruscal-Wallistest, Dunn's multiple comparison test). On the other hand, nosignificant difference occurred between groups at each week ofmeasurement.

These findings demonstrated that DGLA has an action of improvingsymptoms of atopic dermatitis and, in particular, it has an itchsuppressing action.

INDUSTRIAL APPLICABILITY

The present invention provides an agent for external use on skin that issafe to use and effective against inflammatory diseases.

1-14. (canceled)
 15. A method of keeping skin in healthy condition,comprising administering topically, to the skin of a human, an effectiveamount of dihomo-γ-linolenic acid (DGLA) as an active ingredient in acosmetic product, wherein the DGLA is provided in the form of an oilderived from a culture of micro-organisms of genus Mortierella, andwherein the DGLA is applied continuously for at least 14 consecutivedays to the skin.
 16. The method according to claim 15, wherein thedihomo-γ-linolenic acid (DGLA) is in the form of any one of a glyceride,a phospholipid, a glycolipid, an alkyl ester, a salt, or a free fattyacid.
 17. The method according to claim 16, wherein the glyceride is atriglyceride, a diglyceride, or a monoglyceride.
 18. The methodaccording to claim 16, wherein the alkyl ester is an ester with a lowerC₁₋₄ alcohol.
 19. The method according to claim 16, wherein thephospholipid is diacylglycerophospholipid orlysoacylglycerophospholipid.
 20. The method according to claim 15,wherein the dihomo-γ-linolenic acid (DGLA) is administered in acomposition containing 0.01 to 50 wt % of the dihomo-γ-linolenic acid(DGLA).
 21. The method according to claim 14, wherein the DGLA isadministered in the form of an ointment comprising DGLA containing oiland zinc oxide.